Angiogenesis (also called neovascularization), the formation and differentiation of blood vessels from pre-existing vessels or endothelial progenitor cells, is important in both health and disease. Angiogenesis occurs normally during embryogenesis and development, and occurs in fully developed organisms during wound healing and placental development. In addition, angiogenesis occurs in various pathological conditions such as diabetic retinopathy and macular degeneration due to neovascularization, rheumatoid arthritis, inflammatory bowl disease and in cancer, where the newly formed blood vessels supply oxygen and nutrients to the growing tumor. Endothelial cells lining the mature blood vessels normally do not proliferate. During vascular remodeling and angiogenesis, endothelial cells show increased expression of cell surface molecules that potentiate cell invasion and proliferation. Integrins, the super family of heterodimeric membrane bound proteins consisting of several different alpha and beta subunits, are one such class of several cell surface molecules which are up regulated in the proliferating endothelial cells of the tumor vasculature and are also found on different tumor cells including metastatic melanoma cells.
Integrins are important for attachment of cells to the extracellular matrix; cell-cell interactions and signal transduction. A large number of pathogens including Typanosoma cruzi, adenovirus, echovirus, foot-and-mouth disease virus as well as the enteropathogen Y. pseudotuberculosis pathogens exploit integrin receptors for entering our body cells. A distinguished advantage of the integrin-mediated internalization process is that it proceeds by a phagocytic-like process allowing internalization of relatively large structures such as pathogenic bacteria with diameters as big as one to two micrometers. In other words, integrin-specific targeting of non-viral vectors holds potential in avoiding the lower size limitation imposed by the clathrin-coated vesicles commonly involved in many other receptor-mediated endocytosis. Interestingly, the amino acid sequence arginine-glycine-aspartic acid (RGD) is the most evolutionary conserved feature of many, but not all, natural integrin-binding ligands such as extracellular matrix proteins and viral capsids. Because of their high integrin-receptor affinities, cyclic peptides containing RGD domains have been found to be particularly suitable as integrin-targeting vectors. This is why RGD-ligand mediated targeting of anti-cancer genes/drugs to the integrin receptors over expressed on the endothelial cell surfaces of tumor vasculatures (rather than targeting the tumor cells themselves) is a promising anti-angiogenic approach to combat cancer. To this end, the present invention relates to development of a novel series of simple RGD-lipopeptides with remarkably selective efficacies for delivering genes to cells containing over expressed integrin receptors.
A large number of integrin receptor targeting linear and cyclic peptide ligands have been reported including sequences derived from natural protein ligands or sequences selected from phage display libraries (Pasqualini et al. Nature Biotechnology 1997; 15; 542-546, Wickham et al. J. Virology 1997; 71; 8221-8229, Arap et al. Science 1998; 279; 377-380, Erbacher et al. Gene Ther 1999; 6; 138-145, DeNardo et al. Cancer Biother. Radiopharm 2000; 15; 71-79, Müller et al. Cancer Gene Ther 2001; 8; 107-117, Janssen et al. Cancer Res 2002; 62; 6146-6151, Schraa, et al. Int. J. Cancer 2002; 102; 469-475). One of the most potent RGD peptides (RGD4C; CDCRGDCFC) is composed of a central RGD motif structurally stabilized by two disulfide bonds (Koivunen et al. Biotechnology 1995; 13; 265-270). Previously, Hart et al. demonstrated that multiple copies of a cyclic RGD-peptides displayed in the major coat protein subunit of fd filamentous phage particles, approximately 900 nm in length, are internalized efficiently by cells in tissue culture in an integrin-mediated manner (Hart et al. J. Biol. Chem. 1994; 269; 12468-12474). Hart et al. also succeeded in exploiting poly-lysinated peptides covalently linked to multiple RGD-peptides for ensuring integrin-mediated gene expression in epithelial cell lines (Hart et al. Gene Ther 1995; 2; 552-554, 1996, WO96/15811). More recently, Hart et al. disclosed that inclusion of a lipid component in the oligolysine/RGD-peptide/DNA complex remarkably improves levels of gene transfection (U.S. Pat. No. 6,458,026, Hart et al. 2002). Very recently, using phage display technology, Holig et al. (Holig, P. et al. Prot. Eng. Design & Selection, 2005; 17; 433-441) has succeeded in isolating a series of novel RGD-lipopeptides which upon incorporation into liposomes exhibited specific and efficient binding to integrin-expressing cells.
Anticancer drug developments have mostly been centered around designing selective antagonists of αvβ3 and αvβ5 integrins (Richard O. Hynes. Nature Med 2002; 8; 918-921, Brooks et al. Science 1994; 264; 569-571, Brooks et al. Cell 1994; 79; 1157-1164, Brooks et al. J. Clin. Invest. 1995; 96; 1815-1822; Friedlander et al. Science 1995; 270; 1500-1502, Friedlander et al. Proc. Natl. Acad. Sci. USA 1996; 93; 9764-9769; Hammes et al. Nature Med 1996; 2; 529-533). However, it has been demonstrated that mice lacking one or both of the αvβ3 and αvβ5 integrins show enhanced tumor growth and extensive angiogenesis (Reynolds et al. Nature Med. 2002; 8; 27-34, Bader et al. Cell 1998; 95; 507-519). Among the 24 different integrin receptors, the fibronectin receptor α5β1 appears to be the most unambiguous, proangiogenic integrin receptor (Anne et al. Cancer Research 2006; 66; 6002-6007, Curley et al. Cell Mol. Life Sci. 1999; 56; 427-441, Francis et al. Arterioscler Thromb Vasc Biol 2002; 22; 927-933). α5β1 integrin receptors are overexpressed in angiogenic endothelial cells and therefore, are likely to represent a new target in antiangiogenic cancer therapy (Kita et al. Cancer Research 2001; 61; 7985-7991). Designs on sterically stealth liposomes containing covalently grafted RGD-functionalities (Xiao-Bing Xiong et al. Pharmaceutical Research 2005; 22; 933-939) and cationic polymer based non-viral gene delivery systems containing RGD-functionalities (Pascale Belguise-Valladier et al. Cytotechnology 2001; 35; 197-201) have been reported previously. However, none of these priorly reported gene transfer reagents exhibited any α5β1 integrin receptor specificity. To this end, the present invention relates to development of a novel series of simple RGD-lipopeptides for α5β1 integrin receptor mediated delivery of genes/drugs.
The present invention relates to development of a novel series of lipopeptides containing integrin-binding simple RGD tripeptide functionalities. The gene transfer properties of this new class of RGD-lipopeptide described herein is remarkably cell tropic for transformed mouse sarcoma cells (L27 cells). The gene transfer efficacies of the RGD-lipopeptides disclosed herein are significantly inhibited when cells are pre-incubated with commercially available integrin-binding cyclic peptide (RGDfV) indicating that the gene transfer process is mediated through integrin receptors. Thus, the present class of RGD-lipopeptides is likely to find future applications in anti-angiogenic cancer therapy for targeting anti-cancer genes/drugs to tumor vasculatures with over expressed integrin receptors.